Here we propose to develop a high-resolution optical molecular imaging system capable of directly imaging a large pool of endogenous biomolecular species, which are currently inaccessible to high-resolution volumetric imaging. We plan to build on our previous success in integrating pump-probe absorption spectroscopy with optical coherence tomography to develop pump-probe optical coherence microscopy (PPOCM). The resulting imaging system will have subcellular resolution with a sensitivity of 105 dB and a corresponding minimum detectable concentration of 1 [unreadable]M of cytochrome c assuming a -40 dB reflector. We expect the tissue penetration depth to be comparable to standard optical coherence microscopy, which is >350 [unreadable]m in oral epithelium at 850 nm. In addition to the molecular image the system will simultaneously acquire the optical coherence microscopy image depicting tissue ultrastructure. We will initially target the ubiquitous heme proteins, which play an important role in numerous physiological processes including oxygen transport (hemoglobin, myoglobin), cellular metabolism (cytochrome c) and drug metabolism (cytochrome P450). Many of the heme proteins (including those listed) exist in sufficient local concentrations to enable detection with PPOCM. In addition, their rich absorption spectra change significantly between oxidized and reduced forms. This will enable the measurement of relative concentrations (oxid/red) leading to direct measures of blood oxygen saturation, cellular metabolism, and drug metabolism. PUBLIC HEALTH RELEVANCE: We believe the technological advances proposed here will provide an invaluable tool for the in vivo measure of biochemical concentration and dynamics in cells and small animals with subcellular resolution. Further research and development utilizing the developed tools may lead to advances in the understanding of the origins and treatment of human disease as well the development of noninvasive and minimally invasive diagnostics in humans. Since the proposed technique does not require tagging of target molecules either chemically or genetically it has a strong potential for dramatically extending the impact of optical molecular imaging in both the clinical and research environment.